Thermodynamic parameters of opioid binding in the presence and absence of G-protein coupling

We have investigated the thermodynamic parameters of various opioid ligands interacting with their receptors in rat brain membranes. Affinity constants (Ka), enthalpy and entropy values were obtained from homologous displacement experiments performed at 0, 24 and 33 degrees C. It was found that all the opioid agonists tested ([3H]dihydromorphine (DHM) mu alkaloid; [3H]DAMGO mu peptide; [3H]deltorphin-B delta peptide) display endothermic binding accompanied with a large entropy increase, regardless of their chemical structure (alkaloid or peptide), or of their mu or delta receptor selectivity. In contrast, binding of the antagonist naloxone is exothermic, mainly enthalpy driven. Na+ or Mg2+ results only in quantitative changes of the thermodynamic parameters. In the presence of the GTP-analog Gpp(NH)p; or Gpp(NH)p + Na+; or Gpp(NH)p + Na- + Mg2+ the affinity of DHM binding dramatically decreases which might reflect functional uncoupling of the receptor-ligand complex and G-proteins. This altered molecular interactions are also indicated by curvilinear van't Hoff plot and entropy increase. It is concluded that the thermodynamic analysis provides means of determining the underlying driving forces of ligand binding and helps to delineate its mechanism.     

Mutation of a highly conserved aspartate residue in the second transmembrane domain of the cannabinoid receptors, CB1 and CB2, disrupts G-protein coupling

The influence of CRF on testosterone production in primary mouse Leydig cell cultures was studied, and the type of CRF receptor (CRF-R) involved in this activity was determined. CRF directly stimulated testosterone production in mouse Leydig cells, but did not influence the maximum human (h)CG-induced testosterone production. The effect was time- and dose-dependent, saturable with an EC50 of 2.84 nM for hCRF, antagonized by the CRF antagonist alpha-helical CRF9-41, and accompanied by intracellular cAMP elevation. The rank order of potency of the natural CRF agonists, hCRF, ovine CRF, sauvagine, and urotensin, corresponded to that of their activities on CRF-R1 in rat pituitary cells and also to that reported for this receptor, but not for CRF-R2, when transfected into various cell lines. Furthermore, the difference in response of mouse Leydig cells to [11-D-Thr,12-D-Phe]- and [13-D-His,14-D-Leu]-ovine CRF corresponded to that measured when COS cells expressing CRF-R1 were activated, but was considerably smaller than that observed for activation of COS cells expressing CRF-R2alpha or -R2beta. The messenger RNA encoding the mouse CRF-R1 was detected by RT-PCR in mouse Leydig cell preparations. In contrast to mouse Leydig cells, CRF agonists had no influence on the basal testosterone and cAMP production by rat Leydig cells, nor did the agonists or antagonist change the hCG-stimulated testosterone and cAMP production by these cells. It is concluded that mouse Leydig cells express CRF-R1, mediating elevation of testosterone production by CRF agonists through cAMP. Because potencies of CRF agonists in activating mouse Leydig cells were more than 10-fold lower compared with their potencies in stimulating rat pituitary cells, it is suggested that the coupling of the CRF-R1 to intracellular signaling in Leydig cells is different from that in corticotropic pituitary cells, at least in quantitative terms.     

G-protein-coupled receptors: molecular mechanisms involved in receptor activation and selectivity of G-protein recognition

Asian students of seven Japanese language schools participated, and data of 292 Chinese, Taiwanese, and Korean students were analyzed in this study. They were asked about (1) attitudes toward their own and other cultures, (2) high regard for their country and culture, (3) self-efficacy and social skills at the moment and when they were in their country, (4) aspired level of social skills in this country, and (5) feeling of adjustment to life in Japan. Main findings were as follows: (1) psychological factors had stronger effects on the feeling than demographic factors. Self-efficacy in particular had a strong effect. (2) Attitudes to own and other cultures were related to self-efficacy and the feeling. (3) Structural analysis revealed a difference in the feeling between students from socialist and capitalist regions. Based on the analysis, a causal model was proposed of psychological and demographic factors leading to feeling of adjustment, and Asian students' adjustment to life in Japan was discussed in terms of adjustment to their inner, psychological environment.     

Palmitoylation: a post-translational modification that regulates signalling from G-protein coupled receptors

This study examines the relationship between fatal breast cancer and use of estrogen replacement therapy (ERT) among women in a large prospective study in the United States. After nine years of follow-up, 1,469 breast cancer deaths were observed in a cohort of 422,373 postmenopausal women who were cancer free at study entry and who supplied information on estrogen use. Results from Cox proportional hazards modeling, adjusted for 11 other potential risk factors, showed that ever-use of ERT was associated with a significantly decreased risk of fatal breast cancer (rate ratio [RR] = 0.84, 95 percent confidence interval [CI] = 0.75-0.94). There was a moderate trend (P = 0.07) of decreasing risk with younger age at first use of ERT. This decreased risk was most pronounced in women who experienced natural menopause before the age of 40 years (RR = 0.59, CI = 0.40-0.87). There was no discernible trend of increasing risk with duration of use in estrogen users at baseline or former users, nor was there any trend in years since last use in former users. The relationship between ERT and breast cancer mortality differed by age at menarche and by a self-reported history of breast cysts. No increased risk of fatal breast cancer with ERT was observed with estrogen use status (baseline/former), age at first use, duration of use, or years since last use. These findings suggest that ever-use of ERT is associated with a 16 percent decreased risk of fatal breast cancer.     

Multiple coupling of human D5 dopamine receptors to guanine nucleotide binding proteins Gs and Gz

We have demonstrated previously that D1 dopamine receptors are coupled to both Gs alpha and Go alpha. We examine here the coupling between human D5 dopamine receptors and G proteins in transfected rat pituitary GH4C1 cells. Similar to D1 receptors, cholera toxin treatment of cells reduced, but did not abolish, D5 agonist high-affinity binding sites, indicating D5 receptors couple to both Gs alpha and cholera toxin-insensitive G proteins. The interaction between D5 receptors and Gs alpha was confirmed by immunoprecipitation studies and by the ability of D5 receptors to stimulate adenylyl cyclase. Unlike D1 receptors, D5 receptors did not display any pertussis toxin-sensitive G-protein coupling to Go alpha or Gi alpha. D5 receptors were also not coupled to Gq alpha and were unable to mediate phosphatidylinositol metabolism. Instead, D5 sites appeared to be coupled to an AIF(-)4-sensitive, N-ethylmaleimide-resistant G protein. Anti-Gz alpha caused immunoprecipitation of 24.2 +/- 5.2% of G protein-associated D5 receptors, indicating coupling between D5 and Gz alpha. The coupling to Gz alpha was specific for D5 receptors, because similar associations were not detected between D1 receptors and Gz alpha.     

High density lipoprotein receptors, binding proteins, and ligands

Several HDL binding proteins, quite disparate in structure, have recently been cloned and their role in HDL metabolism is currently being assessed. High density lipoprotein binding protein, HBP (vigilin), which lacks a transmembrane domain is responsive to cell cholesterol levels, but its physiological significance remains unknown. On the other hand much is known about SR-B1, a member of the class B scavenger receptors. The level of SR-B1 expression correlates with both the selective transfer of cholesteryl ester into cells and cholesterol efflux from cells, the transfers probably mediated after docking of HDL at the cell surface. SR-B1 exhibits broad ligand specificity and, in animal models, appears to be regulated by the action of pituitary hormones that stimulate steroidogenesis, suggesting an important role for steroid hormone production in supplying precursor cholesterol. Another candidate HDL receptor, HB2, one of a pair of liver HDL binding proteins, shows high sequence homology with adhesion molecules, particularly activated leukocyte-cell adhesion molecule (ALCAM). When HB2 is overexpressed in cells, HDL binding increases. After PMA-induced differentiation of monocytes into macrophages, HB2 mRNA is strikingly elevated, which correlates with increased binding of HDL, but is down-regulated by cholesterol loading of macrophages. The ligand specificity of the HDL receptors, confounded by nonspecific lipid interactions, remains controversial. Their affinity for apoA-I versus apoA-I/A-II-rich HDL particles has clinical implications; both specific sequences in apoA-I and amphipathic alpha-helices may determine binding events. Post-receptor-mediated signalling events may regulate cell functions which, although not primarily related to lipid transport, nevertheless protect against coronary artery disease. Growing evidence for the involvement of lipid-poor apoA-I as a mediator of such pathways is also discussed.     

Expression of mannose-ligand receptors on human spermatozoa: effect of lecithin and association with sperm binding to the zona pellucida

OBJECTIVE: To evaluate the change in the expression of mannose-ligand receptors and sperm binding capacity after the incubation of sperm cells with lecithin liposomes. DESIGN: A randomized, blinded-controlled experiment. SETTING: Andrology laboratory at the Lis Maternity Hospital. PATIENT(S): Fifteen fertile sperm donors and 10 subfertile men. INTERVENTION(S): Incubation of sperm samples with either control medium or 1 mg/mL of liposomal lecithin for 2 hours. MAIN OUTCOME MEASURE(S): Expression of mannose-ligand receptors as evaluated by mannosylated bovine serum albumin-fluorescein isothiocyanate and sperm binding to the zona pellucida as evaluated by the hemizona assay. RESULT(S): The mean +/- SE percentages of spermatozoa with patterns I, II, and III were 86% +/- 4.8%, 11% +/- 3.4%, and 3% +/- 1.6%, respectively, after treatment with control medium and 71% +/- 5.7%, 22% +/- 3.5%, and 7% +/- 2.5%, respectively, after treatment with lecithin. The same effect of lecithin was observed in the 10 sperm samples from subfertile men. The mean +/- SE numbers of sperm that bound to hemizonae after treatment with control medium or lecithin were 116 +/- 32.4 and 176 +/- 29.6, respectively. Statistically significant correlations were observed between the shift in patterns II and III and the enhancement of sperm binding after lecithin treatment (r = 0.44 and 0.6, respectively). CONCLUSION(S): Lecithin shifts the expression of mannose-ligand receptors to the capacitated and acrosoine-reacted patterns and enhances the binding capacity of the sperm cells.     

Temporal and spatial expression patterns of two G-protein coupled receptors in Drosophila melanogaster

Mutations of the iduronate-2-sulfatase gene have been identified as responsible of Hunter syndrome or mucopolysaccharidosis type II. About 20% of the patients have deletions of the whole gene or other major structural alterations. The mutations found so far include: 34 missense, 8 nonsense, 11 small deletions from 1 to 3 bp, 2 deletions of 8 pb, 2 insertions of 1 bp and 2 insertions of 14 bp, with most leading to a frameshift and premature chain termination. Also 8 different splice-site mutations leading to insertions or deletions in the mRNA have been tabulated. Knowledge of the primary genetic defect allows insight into genotype-phenotype correlation and allows a better understanding of the structure and function of iduronate-2-sulfatase.     

Transforming growth factor-beta receptors on human endometrial cells: identification of the type I, II, and III receptors and glycosyl-phosphatidylinositol anchored TGF-beta binding proteins

BACKGROUND: Mutation of the p53 tumor suppressor gene is the most commonly found genetic alteration in human cancer. The E6 gene product of human papillomavirus (HPV) 16 and 18 can inactivate the p53 protein by promoting its degradation. Because most HPV-positive cervical carcinoma cell lines contain wild-type p53 whereas HPV-negative cell lines have point mutations in the p53 gene, a major role in the development of HPV-negative cervical cancer has been attributed to p53. Recent studies, however, have observed no consistent presence of p53 mutation in HPV-negative primary cervical carcinomas. The MDM2 oncogene, which forms an autoregulatory loop with the wild-type p53 protein, has been found amplified in a high percentage of human sarcomas, thus abolishing the antiproliferative function of p53. METHODS: Forty-three primary cervical carcinomas and 10 autopsy-derived distant metastases from one patient were examined for p53 mutation and MDM2 amplification. These tumors had been selected from 238 cervical cancers that had been HPV-typed by Southern blot hybridization and polymerase chain reaction as a representative sample for their HPV status and their clinicopathologic characteristics. Seventeen of the cases had a remarkably good or poor clinical outcome. Human papillomavirus DNA sequences had been detected in 30 of these 43 primary tumors and 13 were negative for HPV by both methods. p53 mutation in the highly conserved exons 5-8 was studied by single-strand conformation polymorphism analysis and direct sequencing. MDM2 amplification was analyzed by Southern blot hybridization. RESULTS: Only two missense point mutations and one nucleotide sequence polymorphism were detected: a TAC-->TGC transition in codon 234 in exon 7, resulting in a Tyr-->Lys substitution, a CGT-->TGT transition in codon 273 in exon 8, resulting in an Arg-->Cys substitution and a polymorphism (CGA-->CGG) in codon 213 in exon 6. Both tumors revealing the point mutations were HPV-negative carcinomas. Amplification of the MDM2 gene was observed in 1 of the 53 specimens tested. CONCLUSIONS: In contrast to data derived from cultured cervical carcinoma cell lines and primary sarcomas, these results indicate that p53 mutation and amplification of the MDM2 oncogene are rare even in HPV-negative primary cervical carcinomas. However, to the authors; knowledge, this is the first observation of MDM2 amplification in humans outside sarcomas and neuroepithelial tumors.     

Presence of bacterial binding 'lectin-like' receptors on phagocytes

Lectin-like receptors capable of binding the bacterium Staphylococcus albus have been demonstrated in the membranes of phagocytes including macrophages, neutrophils and eosinophils from various sources and species. Such receptors are likely to contribute to bacterial adherence and phagocytosis in the non-immune animal.     

Scavenger receptors: diverse activities and promiscuous binding of polyanionic ligands

Scavenger receptors are a diverse family of proteins that share a common property--the binding of modified lipoprotein--but they have recently been shown to recognise a diverse range of ligands. Understanding the molecular interaction of receptor-ligand binding should provide insight into how scavenger receptors contribute to important biological processes.     

Comparative analysis of three murine G-protein coupled receptors activated by sphingosine-1-phosphate

The present study examined the lectin labeling of diverse morphological forms of microglia in culture. Similar to amoeboid microglial cells in vivo, polymorphic microglia showed lectin labeling at their plasma membranes, as well as in a few cytoplasmic vesicles and vacuoles. This labeling pattern was observed in cultured microglia incubated with isolectin at 4 degrees C for 30 min. Five minutes after the temperature was raised to 37 degrees C, the surface lectin receptors appeared to be internalized, as shown by the occurrence of many subsurface lectin-labeled vesicles, vacuoles and tubule-like structures. With longer incubation (up to 1-2 h at 37 degrees C), many lysosomes and a few trans-Golgi saccules and associated lysosome-like structures became labeled. Concomitant with these changes was a reduction of lectin labeling at the plasma, with labeling having vanished in most of the cells after 1-2 h of incubation. By 24 h, only a few cells retained surface lectin labeling. It appears, therefore, that irrespective of morphology, lectin labeling (including its intracellular pathway) of microglia in culture parallels that of amoeboid microglia in vivo. This would offer a useful model for the study of lectin turnover in microglia and help to explain the roles of such receptors in microglial differentiation and function.     

Expression, characterization, and purification of C-terminally hexahistidine-tagged thromboxane A2 receptors

Thromboxane A2 (TxA2) receptors belong to the class of G-protein-coupled receptors. Knowledge of the relationship of structure to function for TxA2 receptors is limited because of their low levels of expression, lengthy purification procedures and poor recoveries. A C-terminal hexahistidine-tag (C-His) was ligated to the alpha-isoform of TxA2 receptors and expressed in COS-7 and Chinese hamster ovary cells. The C-His-TxA2 receptors bound the radioligands 125I-7-[(1R,2S,3S,5R)-6, 6-dimethyl-3-(4-benzenesulfonylamino)bicyclo[3.1. 1]hept-2-yl]-5(Z)-heptenoic acid, an antagonist, and 125I-[1S-1alpha, 2beta(5Z),3alpha(1E,3S*), 4alpha]-7-[3[(3-hydroxy-4-(4'-phenoxy)-1butenyl)-7-oxabicycl o-[2.2. 1]heptan-2-yl]-5-heptanoic acid, an agonist, with affinities not significantly different from those of the wild type (wt)-TxA2 receptors. LipofectAMINE transfection of the cDNAs resulted in high levels of expression (Bmax = 95 +/- 6 pmol/mg) of the C-His-TxA2 receptors. In competition binding studies the IC50 values of five different ligands were not significantly different between C-His-TxA2 and wt-TxA2 receptors. Agonist-induced stimulation of cAMP and total inositol phosphate formation were not significantly different between the two receptors. Purification on a Ni2+-NTA column resulted in a rapid (within 4 h) purification with a 36 +/- 2% recovery and a 30 +/- 6-fold purification (n = 5). The partially purified receptors were resolved on SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, dissolved in acetone/trifluoroacetic acid/hexafluoroisopropanol/sinapinic acid, and successfully subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. The results suggest that the combination of a high level of expression of C-His-TxA2 receptors and a rapid purification procedure followed by SDS- polyacrylamide gel electrophoresis may provide a useful approach for mass-spectrometry based structure-function and other studies of TxA2 receptors.     

Adrenergic receptors in premenstrual dysphoric disorder: I. Platelet alpha 2 receptors: Gi protein coupling, phase of menstrual cycle, and prediction of luteal phase symptom severity

BACKGROUND: Abnormal alpha 2-adrenergic receptor (AR) function is implicated in anxiety and depressive disorders. Premenstrual dysphoric disorder (PMDD) is characterized by anxiety and depressive symptoms, which may be associated with changes in alpha 2AR function. Previous studies on alpha 2AR function during phases of the menstrual cycle in controls and PMDD patients are inconsistent. METHODS: alpha 2AR function was examined in 16 PMDD patients and 15 controls during the follicular phase, and in 10 PMDD patients during late luteal phase. Antagonist-measured maximum binding capacity, agonist-measured receptor density in high- and low-conformational states, and agonist affinity to both states were measured. Coupling efficiency to Gi protein was estimated. RESULTS: There were no significant differences in coupling efficiency. PMDD patients had significantly low antagonist affinity; there were no differences in other binding parameters. There were no changes in alpha 2AR binding parameters between phases of menstrual cycle in PMDD women. alpha 2AR density and symptom severity were inversely related during the follicular phase in controls and patients. During luteal phase, alpha 2AR density correlated positively with symptom severity in patients. High follicular alpha 2AR density predicted more severe luteal symptoms in PMDD patients. CONCLUSIONS: These findings are discussed in view of the molecular biology of alpha 2AR, and their role in PMDD, anxiety, and depressive disorders.     

Insulin-like growth factor receptors and binding proteins

The insulin-like growth factor receptors are integral membrane proteins and demonstrate separate, but important effects on the regulation of cellular processes. The IGF-I receptor signals multiple cascades via its inherent tyrosine kinase activity. The IGF-II/M-6-P receptor on the other hand is primarily involved in targeting of enzymes to various subcellular compartments. In contrast, the insulin-like binding proteins are secreted by the cells and accumulate in the extracellular matrix or on the external surface of the cell. They are also involved in regulating cellular processes more indirectly. They modulate the interactions of the IGFs with their receptors, and in addition, may have some IGF-independent effects probably by direct interaction with integrin and other cell membrane receptor proteins. The recent studies, as outlined in this review, strongly suggest an important, if not essential role for the IGF system in normal physiology and disease states. The challenge now is to define the mechanisms involved in these effects. More studies are required to fully understand the post-receptor mechanism involved in IGF-I receptor signal transduction and the mechanisms whereby the IGFBPs exert their interesting effects. Understanding these mechanisms will enable investigators to create new therapeutic modalities for diseases that are affected by the IGF system.     

Gastroprotective actions of bombesin, L-DOPA, and mild irritants: roles of prostaglandins and sensory neurons

BACKGROUND: Bombesin and dopamine prevent gastric injury by an unknown mechanism. Sensory neurons and endogenous prostaglandins play an important role in gastric mucosal defense. This study was designed to assess the role of these two local defense mechanisms in bombesin and dopamine-induced gastroprotection, as well as mild irritant-induced adaptive cytoprotection. METHODS: Conscious, fasted rats were given either capsaicin (125 mg/kg subcutaneously) to ablate sensory neurons or indomethacin (5 mg/kg intraperitoneally) to inhibit prostaglandin synthesis, 2 weeks and 30 minutes, respectively, before administration of bombesin (100 micrograms/kg subcutaneously), the dopamine precursor L-DOPA (25 mg/kg intraperitoneally), or the mild irritant 25% ethanol (1 mL orogastric). A 1-mL orogastric bolus of acidified ethanol (150 mmol/L HCl/50% ethanol) was given 30 minutes after pretreatment with these peptides and 15 minutes after administration of the mild irritant. Rats were killed 5 minutes later and the total area of macroscopic gastric injury quantified. RESULTS: Ablation of sensory neurons with capsaicin negated the protective actions of bombesin but failed to reverse gastroprotection by L-DOPA or 25% ethanol. Cyclooxygenase inhibition with indomethacin partially reversed bombesin and mild irritant-induced gastroprotection but did not diminish the protective actions of L-DOPA. CONCLUSIONS: Bombesin requires intact sensory neurons to exert its protective actions through a mechanism mediated, at least in part, by endogenous prostaglandins. Adaptive cytoprotection by the mild irritant 25% ethanol requires the presence of endogenous prostaglandins but not sensory neurons. L-DOPA-induced gastroprotection is independent of both local defense mechanisms.     

Monopoly money: The effect of payment coupling and form on spending behavior.

This article examines consumer spending as a function of payment mode both when the modes differ in terms of payment coupling (association between purchase decision and actual parting of money) and physical form as well as when the modes differ only in terms of form. Study 1 demonstrates that consumers are willing to spend more when a credit card logo is present versus absent. Study 2 shows that the credit card effect can be attenuated when people estimate their expenses using a decomposition strategy (vs. a holistic one). Noting that credit card and cash payments differ in terms of payment coupling and form, Studies 3 and 4 examine consumer spending when the payment mode differs only in physical form. Study 3 demonstrates that consumers spend more when they are spending scrip (a form of stored value certificate) versus cash of the same face value. Study 4 shows that the difference in spending across payment modes (cash and gift certificates) is attenuated by altering the salience of parting with money through contextual manipulations of the differences between cash and gift certificates. (PsycINFO Database Record (c) 2010 APA, all rights reserved)